Elucidating Human Mitosis Using an Anaphase-Like Cell-Free System.
Elucidating Human Mitosis Using an Anaphase-Like Cell-Free System.
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DOI：
10.1007/978-1-0716-1538-6_11
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Tzur A
中科院分区：
文献类型：
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作者：
Wasserman D;Nachum S;Noach-Hirsh M;Auerbach N;Sheinberger-Chorni E;Enrico TP;Lahmi R;Emanuele MJ;Tzur A
通过有丝分裂和细胞分裂的平衡进展在很大程度上依赖于有序的磷酸化和泛素介导的调节和结构蛋白的蛋白水解。这一系列事件最终确保了细胞增殖过程中基因组的稳定性和时不变的细胞特性。在所有真核生物中调节有丝分裂里程碑的两种核心酶是细胞周期蛋白依赖性激酶1（CDK 1）及其共激活因子细胞周期蛋白B和E3泛素连接酶后期促进复合物/细胞周期体（APC/C）。发现这些酶的机制和底物对于理解细胞如何通过有丝分裂和高保真分离染色体至关重要。然而，这些酶的研究具有重大挑战。体外研究低估了尚未描述的调节因子的作用，并且错过了细胞环境的生理背景。体内研究是复杂的，因为这些酶中的每一种，以及它们的许多调节剂和下游靶点都是必不可少的。此外，长期的体内操作可能导致级联的间接效应，从而扭曲数据分析和解释。这些挑战中的许多可以使用无细胞系统来规避，无细胞系统在历史上在确定这些酶及其在准细胞环境下的贡献方面发挥了关键作用。在这里，我们描述了一个新开发的人类无细胞系统，重演人类细胞的后期样状态的准备。这个新的工具包补充了来自人类细胞和青蛙卵的传统无细胞系统，可以在细胞生物学实验室中轻松实现，用于磷酸化，APC/C介导的蛋白水解等调节的有丝分裂信号的直接和定量研究。
A balanced progression through mitosis and cell division is largely dependent on orderly phosphorylation and ubiquitin-mediated proteolysis of regulatory and structural proteins. These series of events ultimately secure genome stability and time-invariant cellular properties during cell proliferation. Two of the core enzymes regulating mitotic milestones in all eukaryotes are cyclin dependent kinase 1 (CDK1) with its coactivator cyclin B, and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Discovering mechanisms and substrates for these enzymes is vital to understanding how cells move through mitosis and segregate chromosomes with high fidelity. However, the study of these enzymes has significant challenges. Purely in vitro studies discount the contributions of yet to be described regulators and misses the physiological context of cellular environment. In vivo studies are complicated by the fact that each of these enzymes, as well as many of their regulators and downstream targets, are essential. Moreover, long-term in vivo manipulations can result in cascading, indirect effects that can distort data analysis and interpretation. Many of these challenges can be circumvented using cell-free systems, which have historically played a critical role in identifying these enzymes and their contributions under quasicellular environments. Here, we describe the preparation of a newly developed human cell-free system that recapitulates an anaphase-like state of human cells. This new toolkit complements traditional cell-free systems from human cells and frog eggs and can be easily implemented in cell biology labs for direct and quantitative studies of mitotic signaling regulated by phosphorylation, APC/C-mediated proteolysis, and beyond.